G protein-coupled receptors (GPCRs) are the largest integral membrane protein family in the human genome. These receptors function to transduce signals across the plasma membrane allowing cells to respond and adapt to a variety of different stimuli, such as light, odorants, hormones and many other signaling molecules. This diverse family of receptors is intimately associated with a variety of disease states, including cancer and diabetes, making them attractive targets for drug therapy; it is estimated that 30-50% of prescribed medications target pharmaceutically relevant GPCRs. The aim of this proposal is to identify intracellular proteins that regulate and modulate Ste2p, a yeast receptor that has been used widely as a model for GPCR structure and function. We propose to bridge a large gap in our knowledge of the initial events of GPCR-mediated signal transduction by identifying the quiesome (proteins associated with the receptor in the quiescent or resting state) and the signalosome (intracellular proteins which are recruited and interact with GPCRs upon activation). To accomplish these aims, we will utilize unnatural amino acid replacement to genetically modify Ste2p to contain benzoylphenylalanine (Bpa), a photoactivatable, unnatural amino acid to allow the capture of proteins associated with the intracellular domains of Ste2p in intact, living cells in the presence and absence of its peptide ligand. Our hypothesis is that specific proteins interact with intracellular domains of Ste2p, that these proteins can be captured by the use of laser activation of Bpa in order to generate a time-resolved picture of the intracellular protein partners which will be identified by mass spectrometry, and that our methods using live cells, laser activation will yield a more accurate and dynamic account of GPCR protein partners compared to other methods used to date such as immunoprecipitation. It is expected that the experimental techniques proposed herein will be applicable to medically important GPCRs and that the description of the quiesome and signalosome will provide new targets for drug design leading to new and effective therapies for diseases involving GPCRs.